Bottom Line:
They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents.Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition.They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

ABSTRACTCyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K(+) channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K(+) channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

pone-0001332-g005: M-channel opener properties of compound 15 on Kv7.2/3 channels expressed in CHO cells.(A) Representative traces recorded from the same cell before (left panel) and after (right panel) external application 50 µM compound 15. The membrane potential was stepped from −90 mV (holding potential) to +10 mV for 1.5 s pulse duration in 10 mV increments, followed by a repolarizing step to −60 mV. (B) and (C) Cells were stepped from −90 mV to −50 mV every 30 sec for 1.5 sec pulse duration. Current traces were recorded from the same cell in the absence (control) and presence of 10 µM (B) and 50 µM (c) compound 15. (D) The percentage of the current recorded at −50 mV is shown in the presence of 10 µM and 50 µM compound 15 or in its absence, the latter being the control of 100% (n = 10; * p<0.01). (E) The normalized conductance (G/Gmax) was plotted as a function of the test voltages, for control (open squares), 10 µM (solid squares) and 50 µM (diamonds) compound 15-treated cells. The activation curves were fitted using one Boltzmann function (n = 5). (F) The potency of compound 15 was determined by the extent of left-shift (ΔV50), plotted as a function of compound 15 concentration and fitted by a sigmoidal function yielding an EC50 value of 22±1 µM (n = 5).

Mentions:
As described above, we found that the ester compound 15 of the diclofenac series was both an inhibitor of the COX enzymes but also a potent opener of M-channels. We analyzed in more details its opener properties in vitro and in vivo. Figure 5A (left panel) shows representative traces of Kv7.2/3 channels expressed in CHO cells. External application of compound 15 enhanced Kv7.2/3 currents at nearly all voltages (right panel). However, like all openers described in this study, the effects of compound 15 were voltage-dependent. As the test potentials were more positive (above -10 mV), the effects of compound 15 became weaker on current amplitude. When membrane potential was stepped every 30 sec from −90 to −50 mV, application of 10 µM and 50 µM of compound 15 produced 3.2- and 9.1-fold increase in current amplitude, respectively (n = 10; Figure 5B–D). The drug action had a fast onset as within one minute of compound 15 superfusion, the current increased significantly. When we analyzed the conductance/voltage relationships, it was clear that the activating effect of compound 15 mainly arises from a left-shift in the Kv7.2/3 activation curve. For example, 10 µM, 50 µM and 200 µM of compound 15 respectively produced left-shifts of −11 mV, −21 mV and −31 mV compared to control, with no change in the Boltzmann slopes (Figure 5E and F; V50 = −32.1±8.9 mV, n = 19; V50 = −43.6±9.0 mV, n = 5; V50 = −53.5±9.3 mV, n = 5 and V50 = −63.1±2.0 mV, n = 5, respectively). The opener action of compound 15 was concentration-dependent and was quantified by the extent of the left-shift (ΔV50), yielding an EC50 of 22 µM. Compound 15 also affected Kv7.2/3 gating by accelerating the activation kinetics (from t1/2 = 296±63 ms to t1/2 = 171±29 ms, n = 6; p<0.05; Figure 6A and B). Comparison of the tail currents at −60 mV revealed that compound 15 markedly slowed down the deactivation kinetics of Kv7.2/3 channels (from τdeact = 129±22 ms to τdeact = 284±43 ms; n = 5, p<0.01; Figure 6C and D).

pone-0001332-g005: M-channel opener properties of compound 15 on Kv7.2/3 channels expressed in CHO cells.(A) Representative traces recorded from the same cell before (left panel) and after (right panel) external application 50 µM compound 15. The membrane potential was stepped from −90 mV (holding potential) to +10 mV for 1.5 s pulse duration in 10 mV increments, followed by a repolarizing step to −60 mV. (B) and (C) Cells were stepped from −90 mV to −50 mV every 30 sec for 1.5 sec pulse duration. Current traces were recorded from the same cell in the absence (control) and presence of 10 µM (B) and 50 µM (c) compound 15. (D) The percentage of the current recorded at −50 mV is shown in the presence of 10 µM and 50 µM compound 15 or in its absence, the latter being the control of 100% (n = 10; * p<0.01). (E) The normalized conductance (G/Gmax) was plotted as a function of the test voltages, for control (open squares), 10 µM (solid squares) and 50 µM (diamonds) compound 15-treated cells. The activation curves were fitted using one Boltzmann function (n = 5). (F) The potency of compound 15 was determined by the extent of left-shift (ΔV50), plotted as a function of compound 15 concentration and fitted by a sigmoidal function yielding an EC50 value of 22±1 µM (n = 5).

Mentions:
As described above, we found that the ester compound 15 of the diclofenac series was both an inhibitor of the COX enzymes but also a potent opener of M-channels. We analyzed in more details its opener properties in vitro and in vivo. Figure 5A (left panel) shows representative traces of Kv7.2/3 channels expressed in CHO cells. External application of compound 15 enhanced Kv7.2/3 currents at nearly all voltages (right panel). However, like all openers described in this study, the effects of compound 15 were voltage-dependent. As the test potentials were more positive (above -10 mV), the effects of compound 15 became weaker on current amplitude. When membrane potential was stepped every 30 sec from −90 to −50 mV, application of 10 µM and 50 µM of compound 15 produced 3.2- and 9.1-fold increase in current amplitude, respectively (n = 10; Figure 5B–D). The drug action had a fast onset as within one minute of compound 15 superfusion, the current increased significantly. When we analyzed the conductance/voltage relationships, it was clear that the activating effect of compound 15 mainly arises from a left-shift in the Kv7.2/3 activation curve. For example, 10 µM, 50 µM and 200 µM of compound 15 respectively produced left-shifts of −11 mV, −21 mV and −31 mV compared to control, with no change in the Boltzmann slopes (Figure 5E and F; V50 = −32.1±8.9 mV, n = 19; V50 = −43.6±9.0 mV, n = 5; V50 = −53.5±9.3 mV, n = 5 and V50 = −63.1±2.0 mV, n = 5, respectively). The opener action of compound 15 was concentration-dependent and was quantified by the extent of the left-shift (ΔV50), yielding an EC50 of 22 µM. Compound 15 also affected Kv7.2/3 gating by accelerating the activation kinetics (from t1/2 = 296±63 ms to t1/2 = 171±29 ms, n = 6; p<0.05; Figure 6A and B). Comparison of the tail currents at −60 mV revealed that compound 15 markedly slowed down the deactivation kinetics of Kv7.2/3 channels (from τdeact = 129±22 ms to τdeact = 284±43 ms; n = 5, p<0.01; Figure 6C and D).

Bottom Line:
They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents.Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition.They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

ABSTRACTCyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K(+) channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K(+) channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.